Utilization of Whole Exome Sequencing in Lethal Form of Multiple Pterygium Syndrome: Identification of Mutations in Embryonal Subunit of Acetylcholine Receptor

The acetylcholine receptor (AChR) is a member of the superfamily of transmitter-gated ion channels having a critical role in controlling electrical signals between nerves and muscle cells. Disruptive mutations in genes encoding different subunits of AChR result in multiple pterygium syndrome (MPS), which can be associated with a severe prenatally lethal presentation. This study aimed to investigate the etiology of lethal MPS (LMPS) in two consanguineous families with a history of miscarriages. DNA was extracted from a tissue sample of two aborted fetuses (probands) from two different families with a history of spontaneous miscarriages. Parental peripheral blood samples were collected for confirmatory analysis and follow-up testing. Whole-exome sequencing (WES) was performed on DNA from the probands. The results were confirmed and segregated by Sanger sequencing. Moreover, protein structure evaluations were accomplished. We identified a homozygous frameshift mutation of c.753_754delCT (p.V253fs*44) and a homozygous missense mutation of c.715C>T (p.Arg239Cys) in the CHRNG gene. Both aborted fetuses had pterygium, severe arthrogryposis, and fetal hydrops with cystic hygroma, being compatible with LMPS. The heterozygous state was confirmed in parents for both CHRNG variants. Likewise, CHRNG mutation was predicted to display the damaging effects by lowering the number of helixes and modifying the surface electrostatic potential. The present study identified rare sequence variants in the CHRNG gene in aborted fetuses from consanguineous couples with recurrent miscarriage history. WES is a comprehensive and cost-effective approach to study heterogeneous diseases including MPS. Such findings can improve our knowledge of MPS databases, particularly for genetic counseling of high-risk families and preimplantation genetic diagnosis.

Fetuses with this condition are usually not born.
Although autosomal recessive inheritance appears to be the most common form of this disease (3,4), X-linked and autosomal dominant inheritance patterns are, also, observed in a few cases (5).
A huge body of literature represents that mutations in the cholinergic receptor nicotinic gamma subunit (CHRNG) gene lead to autosomal recessive MPS (6).The acetylcholine receptor (AChR), playing role as an excitatory cation channel, is a member of the superfamily of transmitter-gated ion channels with a critical role in controlling electrical signals between nerves and skeletal muscle cells acting through opening and closing a membrane-spanning pore (7). In humans, AChR is a transmembrane pentameric glycoprotein having four different types of subunits including two α (CHRNA1), one β (CHRNB), one δ (CHRND), and one γ/ε (CHRNG/CHRNE) subunits.
Interestingly, the AChR is available in two forms namely embryonic, which is present in fetal and denervated muscle, and the adult, predominantly expressed after birth. At the 33 rd week of gestation, the embryonic AChR is switched to the adult type (8,9). Worthy of note, the CHRNG gene, encoding the γ subunit of AChR, is merely expressed during early fetal development, while it is replaced by CHRNE, encoding ε subunit, in adult humans.  agarose gel-electrophoresis.

Mutations in the
The WES was used on DNA from the proband.
As the next step, DNA was fragmented and the targeted enrichment was done using the SureSelect  (12,13).

Tertiary structure prediction and validation
The three-dimensional structural predictions of ACHG and its mutant protein were generated using the iterative threading assembly refinement (I-TASSER) server (14). The top ranking models were refined by GalaxyRefine server (15). The quantitative evaluation of selected models were carried out using VADAR and ProSA web tools (16,17).

Visual presentation
All protein figures were created using PyMOL software (18).  Table 2, the most second structures were random coils for both native and mutant protein; however, helixes are more predicted in native structure.    The five predicted models of tertiary structure of the native and mutant protein were obtained by I-TASSER server and the best models were model 1

WES findings
with the best C-score. The C-score, TM score and

Discussion
In  Table 4 and Figure7.
AChR is a member of transmitter gated ion channel superfamily. Two forms of skeletal muscular AChR are found in mammalian and identified by their different function and subunit compositions (19). Gamma subunit is expressed in fetus and epsilon is expressed in mature skeleton.
Fetal AChR type is present before week 33 of gestation in human, and is gradually replaced with the epsilon subunit mediated by acetylcholine receptor-inducing activity (ARIA) (20)(21)(22). Before the insertion of receptor into the membrane, the five subunits of AChR should be assembled in the endoplasmic reticulum (ER) (23,24). The interaction network of CHRNG, CHRNA1, and CHRND with associated genes in MPS is shown in Figure 7. The receptor cannot reach the cell surface whether one subunit is missing or due to AChR deficiency (25). It was previously reported that disruptive mutations in different genes encoding